Genome-wide analysis of differentially expressed mRNAs, lncRNAs, and circRNAs in chicken bursae of Fabricius during infection with very virulent infectious bursal disease virus

- Genome-wide analysis of differentially expressed mRNAs, lncRNAs, and circRNAs in chicken bursae of Fabricius during.
- To investigate interactions between IBDV and its host, RNA sequencing was applied to analyze the responses of the differentially expressed transcriptional profiles of BF infected by very virulent IBDV (vvIBDV)..
- We predicted interactions between differentially expressed genes and produced lncRNA-mRNA and circRNA-miRNA-mRNA regulator network..
- Conclusions: The present study identified the expression profiles of mRNAs, lncRNAs, and circRNAs during vvIBDV infection and provides new insights into the pathogenesis of IBDV and antiviral immunity of the host..
- Infectious bursal disease virus (IBDV), a non-enveloped double-stranded RNA virus, is a member of the family Birnaviridae.
- IBDV predominantly targets the precursors of B lymphocytes, particularly those in the bursa of Fabricius (BF), an important immune organ, the infection of which may lead to B lymphocyte depletion and BF deterior- ation [4, 5].
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- based on the location of the transcripts on the reference genome, a transcript length of ≥200 bp, and an exon count of ≥2.
- No intronic lncRNAs were detected in the current study.
- Besides, anchor reads were mapped to the chicken reference genome, 7808 novel circRNAs were identified in the study (Additional file 5.
- Differentially expressed profiles of mRNAs, lncRNAs, and circRNAs.
- 94 were downregulated in the treatment group (Figs.
- Differentially expressed mRNAs, lncRNAs, and circRNAs were used for cluster analysis.
- The expression patterns of the mRNAs, lncRNAs, and circRNAs thus differed between the two groups, suggesting that the differentially expressed genes (DEGs) in the chicken bursa tissue infected with IBDV were significantly different from those in the non- infected tissue..
- To examine the differences in the mRNAs and lncRNAs, genetic structure and sequence conservation was compared, and the distribution of the transcript length of the lncRNAs differed slightly from that of the mRNAs (Fig.
- However, there were fewer exons in the lncRNAs than in the mRNAs (Fig.
- In addition, the open reading frames of the lncRNAs were shorter than those of the mRNAs (Fig.
- Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to analyze the 411 differentially expressed mRNAs and the tar- get genes of the differentially expressed lncRNAs and cir- cRNAs to examine the functions of gene dysregulation during IBDV infection.
- GO enrichment of the biological pro- cesses (BPs), cellular components (CCs), and molecular func- tions (MFs) of the differentially expressed mRNAs, lncRNAs, and circRNAs is shown in Figs.
- Based on the GO analyses of the differentially expressed lncRNA target genes, the most enriched BPs were cellular processes, biological regulation, and single-organism processes.
- GO enrichment analysis was performed for the antisense, cis, and trans roles of the target genes of the lncRNA, showing that target genes were also mainly enriched in cellular pro- cesses, cells, and binding (Additional file 10: Figure S1).
- The circRNA results were consistent with those of the lncRNAs (Fig.
- b CNCI, CPC, and the SwissProt database were used to analyze the coding potential of the novel lncRNAs.
- According to the KEGG analyses of the target genes of the differentially expressed lncRNAs, spliceosome, JAK-STAT signaling pathway, ribosome, and Toll-like receptor signaling pathway were enriched (Fig.
- antisense, cis, and trans-regulation were mainly enriched in the notch signaling pathway, the glycosphingolipid biosynthesis-lacto complement, and spliceosome, re- spectively (Additional file 12: Figure S2).
- In the cir- cRNAs, the MAPK signaling pathway, protein processing in the endoplasmic reticulum, and ubiquitin- mediated proteolysis were identified as predominantly enriched KEGG pathways (Fig.
- In the study, we predicted the potential target genes of lncRNAs and constructed lncRNA-mRNA regulatory networks (Fig.
- In the lncRNA- mRNA network, LOC112532624 (XR with the most significant difference (fold change = 703.3).
- 2 Venn diagram showing the number of overlapping genes in the IBDV-infected group and the control group.
- In the circRNA-miRNA-mRNA network, circRNAs would in- directly regulate 25 chicken mRNAs, such as STAT1/4 and IRF1/7, indicating that these circRNAs might play a critical role in regulating vvIBDV-infection..
- To validate the accuracy of the RNA-sequencing results, 10 differentially expressed mRNAs, lncRNAs, and cir- cRNAs were selected and quantified by reverse- transcription quantitative PCR (RT-qPCR.
- Infectious bursal dis- ease caused by IBDV is one of the most important infec- tious diseases that severely affect the poultry industry..
- 3 Histogram of the differentially expressed mRNAs (a), lncRNAs (b) and circRNAs (c) in the two groups.
- Table 3 The number of differentially expressed mRNA and lncRNA in IBDV treated group.
- predominant clinical disease type in nearly all poultry- producing regions of the world.
- The re- sults indicated that these DEGs may play a significant role in the vvIBDV infection process, suggesting that they may include potential therapeutic targets for treat- ing IBDV infections..
- In our study, the JAK-STAT sig- naling pathway, the NOD-like receptor signaling path- way, the cytokine-cytokine receptor interaction signaling pathway, apoptosis, the chemokine signaling pathway, and the Toll-like receptor signaling pathway were signifi- cantly enriched according to the KEGG enrichment ana- lyses of the respective differentially expressed mRNAs..
- IBDV exploits these pathways to induce the expression of STAT1, STAT3, STAT4, TRIM25, IFIT1, and Mx1 in the bursal tissue, and our results were in line with those of previous studies.
- In STAT1, a member of the STAT family, phosphorylation induces the expression of interferon-stimulating genes through a series of signal transduction steps to elicit antiviral mechanisms [39,.
- 4 Volcano plots of the differentially expressed mRNAs (a), lncRNAs (b), and circRNAs (c).
- 5 Heatmap of differentially expressed mRNAs (a), lncRNAs (b), and circRNAs (c).
- a Number of exons in the mRNAs and lncRNAs.
- b Distribution of transcript lengths in the mRNAs and lncRNAs.
- The horizontal axis indicates the length of the transcripts, and the vertical axis indicates the abundance.
- c Number of open reading frames (ORFs) in the mRNAs and lncRNAs.
- In the current study, it is worth noting that LOC107053928, LOC107054815, LOC107053352, and LOC107053557 were identified as regulated various target genes associated with immunomodulation, includ- ing STAT1, STAT3, STAT4, TRIM25, and IFIH1..
- TRIM25 is a member of the tripartite motif family of E3 ubiquitin ligases and has been demonstrated to play an important role in RIG-I antiviral pathways.
- It has been well established that type I IFNs plays a crucial role in the antiviral processes, where.
- Therefore, this implied that these lncRNAs and their tar- get genes, STAT1, STAT3, STAT4, IFIH1 and TRIM25, might play a vital role in the IBD anti-viral response.
- Therefore, the expression levels of STAT1 and STAT3 may reflect the balanced response of the.
- 7 Gene ontology (GO) analysis of the differentially expressed mRNAs in IBDV-infected chicken BF.
- a-c Directed acyclic graph showing the significantly enriched biological processes, cellular components, and molecular functions of the differentially expressed mRNAs.
- d Number of genes in GO terms are shown in the histogram.
- 8 Gene ontology analysis of the differentially expressed lncRNAs (a) and circRNAs (b) in the two groups.
- In the current study, 63 up- regulated and 80 downregulated circRNAs were identi- fied, and their expression levels were generally lower than those of the mRNAs and lncRNAs.
- In the study, circRNA-miRNA-mRNA network was constructed and showed that the regulatory relationships between cir- cRNAs, miRNAs and target mRNAs were complex.
- In the network, circRNAs novel_circ_000574 and novel_.
- 9 Kyoto Encyclopedia of Genes and Genomes pathway enrichment of the differentially expressed mRNAs (a), lncRNAs (b), and circRNAs (c) in the two groups.
- The dot size indicates the number of DEGs in the pathway, and the coloration corresponds to the Q-value range.
- The red ellipses indicate the differentially expressed lncRNAs, and the green ellipses indicate the differentially expressed mRNAs.
- 12 Validation of the differentially expressed mRNAs (a), lncRNAs (b), and circRNAs (c) by RT-qPCR.
- Chickens in the control group were injected with phosphate-buffered saline (PBS.
- The quality of the libraries was evaluated using a.
- Raw sequencing data were made available in the NCBI short reads archive..
- The intersection of the respective results was chosen as lncRNAs.
- 1.3) [63], phyloFit [64], and PhastCons [65] were used to evaluate sequence conservation in the transcripts, cal- culate phylogenetic models among species, and compute the conservation scores of coding genes and lncRNAs, respectively.
- For circRNA, unmapped reads were ex- tracted from the above results, and the ends of the un- mapped reads were intercepted (default 20 bp) to identify the anchor reads, which were then mapped to the reference genome.
- This software program includes the Vienna RNA package, and best base pairing predic- tions were based on a calculation of minimum free en- ergy in the thermodynamic structure.
- Moreover, the cis and trans target genes of the differentially expressed lncRNAs were predicted.
- For the cis target genes, the mRNAs and the genomic location of the lncRNAs were mapped.
- The differentially expressed lncRNAs (fold change >.
- The binding sites of the miRNAs on the circRNAs and mRNAs were predicted using mirTarBase software [68], and the target relationships of miRNAs-mRNAs and miRNAs-circRNAs were assessed accordingly.
- To understand the functions of the differentially expressed transcripts, including the mRNA and the tar- get genes of the lncRNAs and circRNAs, the GO (http://.
- RT-qPCR validation of the RNA-sequencing results.
- To assess the accuracy of the sequencing results, five up- regulated and five downregulated mRNAs, lncRNAs, and circRNAs were selected and quantified by RT-qPCR..
- β-Actin was used as an internal control of the mRNA.
- The 2 −ΔΔCt method was used to calculate the relative expression levels of the target genes.
- Data are shown as the means ± standard error of the mean.
- All the differentially expressed mRNAs in this study..
- All the differentially expressed lncRNAs in this study..
- All the differentially expressed circRNAs in this study..
- Gene ontology enrichment analysis for the antisense, cis, and trans roles of the differentially expressed lncRNAs in chicken BF between the two groups.
- Kyoto Encyclopedia of Genes and Genomes pathway enrichment for the antisense, cis, and trans roles of the differentially expressed lncRNAs in chicken BF between the two groups.
- The dot size indicates the number of differentially expressed genes in the pathway, and the coloration corresponds to the Q-value range..
- DEGs: Differentially expressed genes.
- These funding agencies provided great support in funding, and played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript..
- The raw data sets supporting the results of this article are available in the NCBI short reads archive and accession number is PRJNA635782.
- recommendations in the institutional and national guidelines for animal care and use.
- Phylogenetic analysis of the polyprotein coding region of an infectious South African bursal disease virus (IBDV) strain.
- Transcriptional profiling reveals a possible role for the timing of the inflammatory response in determining susceptibility to a viral infection.
- Circular transcripts of the testis-determining gene Sry in adult mouse testis.
- Infectious bursal disease virus: strains that differ in virulence differentially modulate the innate immune response to infection in the chicken bursa..
- Transcription profiles of the responses of chicken bursae of Fabricius to IBDV in different timing phases.
- Interactions between STAT and non-STAT proteins in the interferon-stimulated gene factor 3 transcription complex.
- A diverse range of gene products are effectors of the type I interferon antiviral response.
- Long non-coding RNAs in the regulation of the immune response.
- Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway.
- Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease..
- accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions

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